Structurally distinct unfolding intermediates formed from a staphylococcal capsule-producing enzyme retained NADPH binding activity

Abstract

CapF, a capsule-producing enzyme expressed by Staphylococcus aureus, binds NADPH and exists as a dimer in the aqueous solution. Many other capsule-producing virulent bacteria also express CapF orthologs. To understand the folding-unfolding mechanism of S. aureus CapF, herein a recombinant CapF (rCapF) was individually investigated using urea and guanidine hydrochloride (GdnCl). Unfolding of rCapF by both the denaturants was reversible but proceeded via the synthesis of a different number of intermediates. While two dimeric intermediates (rCapF4 and rCapF5) were formed at 0.5 M and 1.5 M GdnCl, three dimeric intermediates (rCapF1, rCapF2, and rCapF3) were produced at 1 M, 2 M, and 3 M urea, respectively. rCapF5 showed 3.6 fold less NADPH binding activity, whereas other intermediates retained full NADPH binding activity. Compared to rCapF, all of the intermediates (except rCapF3) had a compressed shape. Conversely, rCapF3 possessed a native protein-like shape. The maximum shape loss was in rCapF4 though its secondary structure remained unperturbed. Additionally, the tertiary structure and hydrophobic surface area of the intermediates neither matched with each other nor with those of the native rCapF. Of the four Trp residues in rCapF, one or more Trp residues in the intermediates may have higher solvent accessibility. Using sequence alignment and a tertiary structural model of CapF, we have demonstrated that the region around Trp 137 of CapF may be most sensitive to unfolding, whereas the NADPH binding motif carrying region at the N-terminal end of this protein may be resistant to unfolding, particularly at the low denaturant concentrations.

Communicated by Ramaswamy H. Sarma

Keywords:

• CapF
• unfolding
• intermediate
• activity
• molecular modeling

Acknowledgements

We thank Dr. Gopal Chakrabarti (University of Calcutta), Dr. Smarajit Polley (Bose Institute), and Dr. Malabika Biswas (Birla Institute of Technology and Science) for critically reading and rectifying the manuscript. We also thank Mr. S. Biswas, and Mr. M. Das for their excellent technical support. Mr. S. Seal, and Mr. T. Chakraborty received Senior research fellowships from Bose Institute. Ms. D. Sinha received Junior Research Fellowship from Bose Institute. Mr. D. Sinha received Project Assistantship from Bose Institute.

Disclosure statement

No potential conflict of interest was reported by the author(s).